PROJECT SUMMARY Orientia tsutsugamushi (Ot) is a highly virulent, chigger-borne, strictly intracellular Gram-negative bacterium known to cause scrub typhus in humans, a disease endemic to `Tsutsugamushi triangle' placing more than a billion people at risk and responsible for more than a million cases every year. Antigenic and genetic variability among Ot strains is an established phenomenon, yet our appreciation of the regulation of Ot genomes during host-pathogen interactions remains in its infancy. Bacterial small regulatory RNAs (sRNAs) have only recently emerged as critical post-transcriptional regulators of gene expression. Among these, trans-acting sRNAs act by binding to target mRNA(s); cis-acting sRNAs are transcribed antisense to their target RNA; and riboswitches alter gene expression by interacting with either small ligands or metabolites. Small RNAs regulate bacterial virulence by initiation/termination of transcription, stabilization/degradation of target mRNAs, and regulation of translation. Despite their importance as potential therapeutic targets, the identities and functions of Ot sRNAs have remained a mystery. A major bottleneck precluding the exploration of such regulatory networks in Ot pertains to the `sticky' genomes punctuated by extensive homologous recombination driven by transposons, conjugative elements, repetitive sequences, and gene duplication events, but this hurdle can now be overcome based on the sequencing and annotation of six Ot genomes as closed, circular chromosomes, including that of prototypical and highly pathogenic Karp strain (OtK). With a long-term goal of defining sRNA- mRNA interactions in Ot, we have employed this resource for bioinformatic predictions and follow-up validation to identify sRNAs in OtK. Our intriguing preliminary findings suggest that OtK genome does not encode for classical bacterial sRNAs 4.5S and 6S, indicating a unique sRNA repertoire which we hypothesize to play an important role in post-transcriptional gene expression in microvascular endothelium as the preferred, primary target cell niche in mammalian hosts. Accordingly, the objective of this application is to determine the coding and non-coding transcriptomes of OtK in correlation with the corresponding proteome during interactions with the host endothelium. In Aim 1, we will catalogue all expressed sRNAs and coding transcripts and determine their transcriptional start sites during OtK infection of human/mouse endothelial cells by differential RNA sequencing. Aim 2 will then identify and validate cognate mRNA targets for all novel trans- and cis-encoded sRNAs. We will apply a cutting-edge RNA-sequencing platform in conjunction with advanced omics-based approaches and our previously documented experience with the identification and characterization of sRNAs in pathogenic Rickettsia species. The outcomes will positively impact the field via first mechanistic understanding of the contributions of riboregulatory circuitry in Ot to the host-pathogen interactions and pathogenesis of scrub typhus, enabling the discovery of novel targets for the design/development of new and improved therapeutics.